Provided are compounds of the formula (1):

This invention relates to cyclic peptide antifungal agents. In particular, it relates to acyl derivatives of the echinocandin class of cyclic peptide antifungal agents; to methods for treating antifungal and parasitic infections, and to formulations useful in the methods.
The compounds provided by this invention are semi-synthetic antifungal agents in that they are derived from the cyclic peptide antifungals which are produced by culturing various microorganisms. A number of cyclic peptide antifungals are known. Among these are echinocandin B (A30912A), aculeacin, mulundocandin, sporiofungin, L-671,329, FR901379, and S31794/F1. All such antifungals are structurally characterized by a cyclic hexapeptide core, or nucleus, the amino group of one of the cyclic amino acids bearing a fatty acid acyl group forming a side chain off the core or nucleus. For example, echinocandin B has a linoleoyl side chain while aculeacin has a palmitoyl side chain. These fatty acid side chains of the cyclic hexa- peptides can be removed by enzymatic deacylation to provide the free nucleus. (Formula (1), hereinafter, wherein R2 is hydrogen.) Reacylation of the amino group of the nucleus provides semi synthetic antifungal compounds. For example, the echinocandin B nucleus provides a number of antifungal agents when reacylated with certain unnatural side chain moieties (see Debono, U.S. Pat. No. 4,293,489). Among such antifungal compounds is cilofungin which is represented by the formula (1) wherein R is methyl, R1 is hydrogen and R2 is p-(n-octyloxy)benzoyl.
Enzymatic deacylation of the cyclic hexapeptides is carried out with deacylase produced by the organism Actinoplanes utahensis and related microorganisms as described by Abbott et al., U.S. Pat. No. 4,293,482.
The present invention provides acylated cyclic hexapeptides having unique side chain acyl groups which, inter alia impart enhanced antifungal and antiparasitic potency e.g. against pathogenic strains of Candida albicans. Also provided is a process for removing the aminal and benzylic hydroxy groups to result in a dideoxy compound of formula (1) (Rxe2x95x90H).
The compounds provided by this invention are represented by the following formula (1):
wherein
Rxe2x80x2 is hydrogen, methyl or NH2C(O)CH2xe2x80x94;
Rxe2x80x3 and Rxe2x80x3xe2x80x2 are independently methyl or hydrogen;
R and Ry are independently hydroxy or hydrogen;
R1 is hydroxy, hydrogen, or hydroxysulfonyloxy;
R7 is hydroxy, hydrogen, hydroxysulfonyloxy or phosphonooxy; and
I) R2 is a substituted benzoyl group represented by the formula:
xe2x80x83wherein
A) R3 is a polyoxa-alkyl group represented by the formula
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-C12 alkyl)
wherein m and n are integers of from 2 to 4, and p is 0 or 1; or
B) R3 is an unsaturated hydrocarbon group represented by,the formula:
xe2x80x94Yxe2x80x94(C1-C12 alkyl)
wherein Y is xe2x80x94Cxe2x89xa1Cxe2x80x94 or xe2x80x94CHxe2x95x90CHxe2x80x94; or
C) R3 is a group of the formula xe2x80x94Oxe2x80x94(CH2)mxe2x80x94G, wherein m is as defined and G is C7-C10 bicycloalkyl or C7-C14 tricycloalkyl; or
D) R3 is quinolyl; or
II) R2 is an acyl group represented by the formula:
xe2x80x83wherein
Z is xe2x80x94Oxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2xe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94, or a carbon to carbon bond;
A) R4 is hydrogen, C2-C12 alkynyl, C2-C12 substituted alkynyl, C3-C12 cycloalkyl, C7-C10 bicycloalkyl, C7-C14 tricycloalkyl, C1-C12 alkoxy, C3-C12 cycloalkoxy, naphthyl, pyridyl, thienyl, benzothienyl, quinolyl or phenyl; or
B) R4 is phenyl substituted by amino, C1-C12 alkylthio, halogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 substituted alkyl, C2-C12 substituted alkenyl, C2-C12 substituted alkynyl, C1-C12 alkoxy, trifluoromethyl, phenyl, substituted phenyl, phenyl substituted with a polyoxa-alkyl group represented by the formula:
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-C12 alkyl)
wherein m,n and p are as defined; or
C) R4 is phenyl substituted with C1-C6 alkoxy substituted by fluoro, bromo, chloro or iodo; or
D) R4 is C1-C12 alkoxy substituted with C3-C12 cycloalkyl, C7-C10 bicycloalkyl, C7-C14 tricycloalkyl, C2-C12 alkynyl, amino, C1-C4 alkylamino, di-(C1-C4 alkyl)amino, C1-C12 alkanoylamino, phenyl substituted with a polyoxa-alkyl group represented by the formula:
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-C12 alkyl)
wherein m,n and p are as defined; or
E) R4 is C1-C12 alkoxy substituted with a group of the formula:
wherein R8 is C1-C6 alkoxy optionally substituted with phenyl; or
F) R4 is a group represented by the formula:
xe2x80x94Oxe2x80x94(CH2)pxe2x80x2xe2x80x94Wxe2x80x94R5
wherein pxe2x80x2 is an integer of from 2 to 4; W is pyrrolidino, piperidino or piperazino, and R5 is hydrogen, C1-C12 alkyl, C3-C12 cycloalkyl, benzyl or C3-C12 cycloalkylmethyl; or
G) R4 is a group represented by the formula:
xe2x80x94Yxe2x80x94R6
xe2x80x83wherein
Y has the same meanings defined above; and
R6 is C1-C12 alkyl, C1-C12 substituted alkyl; C3-C12 cycloalkyl, C7-C10 bicycloalkyl, C7-C14 tricycloalkyl, phenyl, C3-C12 cycloalkenyl, naphthyl, benzothiazolyl, thienyl, indanyl, fluorenyl, phenyl substituted by amino, C1-C12 alkylthio, halogen, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C12 alkoxy, trifluoromethyl, xe2x80x94Oxe2x80x94(CH2)pxe2x80x2xe2x80x94Wxe2x80x94R5, or C1xe2x80x94C6 alkoxy substituted by fluoro, bromo, iodo or chloro; or
R6 is a phenyl substituted by a polyoxa-alkyl group represented by the formula
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-C12 alkyl)
wherein m,n and p are as defined above; or
III) R2 is a group having the formula:
wherein Rx is C1-C12 alkoxy or a polyoxa-alkyl group represented by the formula:
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-xe2x80x94C12 alkyl)
wherein m,n and p are as defined above; or
IV) R2 is a group having the formula:
wherein R9 is phenyl, C1-C12 alkyl, or C1xe2x80x94C12 alkoxy; or
V) R2 is naphthoyl substituted with R4; and the pharmaceutically acceptable non-toxic salts thereof;
with the proviso that when
Rxe2x80x2 is methyl or NH2C(O)CH2xe2x80x94;
Rxe2x80x3 is methyl;
Rxe2x80x2xe2x80x3 is methyl;
RY is hydroxy;
R is hydroxy; and
either a) or b):
a) R1 is hydroxysulfonyloxy and R7 is hydroxy, hydroxysulfonyloxy or phosphonooxy;
b) R1 is hydrogen or hydroxysulfonyloxy and R7 is hydroxysulfonyloxy or phosphonooxy;
xe2x80x83R2 is not
wherein R3 is
xe2x80x94Oxe2x80x94(CH2)mxe2x80x94[Oxe2x80x94(CH2)n]pxe2x80x94Oxe2x80x94(C1-C12 alkyl)
wherein p=O; nor
wherein Z is a carbon to carbon bond or xe2x80x94Oxe2x80x94 and R4 is C1-C12 alkoxy; nor
iii) naphthoyl substituted by R4 wherein R4 is hydrogen, phenyl, or C1-C12 alkoxy.
Also provided are formulations and methods for inhibiting parasitic and fungal activity which employ the compounds of the invention, and a process for preparing the dideoxy form of the compounds.
The term: xe2x80x9cC1-C12 alkylxe2x80x9d refers to the straight or branched chain alkyl hydrocarbon groups such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl groups; and the like.
The term xe2x80x9cC2-C12 alkenylxe2x80x9d refers to groups such as vinyl, 1-propene-2-yl, 1-butene-4-yl, 1-pentene-5-yl, 1-butene-1-yl, and the like.
The term xe2x80x9cC2-C12 alkynylxe2x80x9d refers to such groups as ethynyl, propynyl, pentynyl, butynyl and the like.
The term xe2x80x9cC1-C12 alkylthioxe2x80x9d refers to such groups as methylthio, ethylthio, t-butylthio, and the like.
The term xe2x80x9cC1-C12 alkoxyxe2x80x9d refers to the straight or branched chain oxyalkyl groups such as, e.g. methoxy, ethoxy, propoxy, butoxy, heptoxy, octyloxy, dodecyloxy, and the like.
The term xe2x80x9cC3-C12 cycloalkoxyxe2x80x9d refers to such groups as cyclopropoxy, cyclobutoxy and the like.
The term xe2x80x9cC3-C12 cycloalkenylxe2x80x9d refers to such groups as cyclopropenyl, cyclobutenyl, cyclopentenyl, and the like.
The term xe2x80x9cC1-C12 substituted alkyl,xe2x80x9d xe2x80x9cC2-Cl2 substituted alkenylxe2x80x9d , and xe2x80x9cC2-C12 substituted alkynylxe2x80x9d, denotes the above substituted one or two times with halogen, hydroxy, protected hydroxy, amino, protected amino, C1-C7 acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carbamoyloxy, cyano, methylsulfonylamino, phenyl, substituted phenyl, or C1-C12 alkoxy.
The term xe2x80x9csubstituted phenylxe2x80x9d is represented by a phenyl group substituted with one, two, or three moieties chosen from halogen, hydroxy, protected hydroxy, cyano, nitro, C1-C12 alkyl, C1-C12 alkoxy, carboxy, protected carboxy, carboxymethyl, hydroxymethoyl, amino, aminomethyl trifluoromethyl or N-(methylsulfonylamino)
The term xe2x80x9cC3-C12 cycloalkylxe2x80x9d refers to such groups as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term xe2x80x9cC1-C4 alkylaminoxe2x80x9d refers to such groups as methylamino, ethylamino, n-butylamino and the like.
The term xe2x80x9cdi-(C1-C4 alkyl)aminoxe2x80x9d refers to such groups as dimethylamino, diethylamino, di-n-propylamino, di-n-butylamino, methylethylamino, methyl-n-butylamino, and like tertiary amino groups.
The term xe2x80x9cC1-C12 alkanoylaminoxe2x80x9d refers to such groups as acylamino groups derived from the C1-C12 carboxylic acids and are exemplified by formamido, acetylamino, propionylamino, butyrylamino, and the like.
The term xe2x80x9cC3-C12 cycloalkylmethylxe2x80x9d refers to those C3-C7 cycloalkyls,described above further substituted by methyl.
The terms xe2x80x9cC7-C10 bicycloalkylxe2x80x9d and xe2x80x9cC7-C14 tricycloalkylxe2x80x9d refer to such groups as bicyclo[2.2.1.]hept-2-yl, bicyclo[2.2.1.]hep-4-en-2-yl, bicyclo[3.3.1.]nona-3-yl, bicyclo[3.3.1.]nona-2-yl, bicyclo[3.2.1.]oct-2-yl, bicyclo[2.2.2.]oct-2-yl, bicyclo[2.2.2]oct-5-en-2-yl, adamantyl and the like.
The term xe2x80x9cdideoxyxe2x80x9d refers to compounds of the formula (1) wherein Rxe2x95x90H.
The term xe2x80x9cinhibitingxe2x80x9d, such as used in relation to the methods for inhibiting parasitic and fungal activity, is defined to mean its normal definition, i.e., to stop, retard or prophylactically hinder or prevent.
The term xe2x80x9cactivityxe2x80x9d, as used in relation to parasitic and fungal activity, includes growth thereof and attending characteristics and results from the existence of the parasite or fungus.
The term xe2x80x9ccontactingxe2x80x9d, as used in relation to the methods for inhibiting parasitic and fungal activity by contacting a compound of the invention with a parasite or fungus, is defined to mean its normal definition. However, the term does not imply any further limitations to the process, such as by mechanism of inhibition, and the methods are defined to encompass the spirit of the invention, which is to inhibit parasitic and fungal activity by the action of the compounds and their inherent anti-parasitic and anti-fungal properties, or in other words, the compounds, used in the method are the causative agent for such inhibition.
Examples of acyl groups represented by R2 in formula (1) are benzoyl substituted by polyoxa-alkyl groups such as, e.g., 2-methoxyethoxy (p=0, m=1), 2-ethoxyethoxy, 2-(2-ethoxyethoxy)ethoxy (m=2, p=1, n=2), 3-(2-ethoxyethoxy)-propoxy, 3-(2-methoxyethoxy)butoxy, and like groups.
Examples of R3 groups wherein R2 is benzoyl substituted by an unsaturated hydrocarbon groups xe2x80x94Yxe2x80x94(C1-C12-alkyl) include e.g., acetylenic groups xe2x80x94Cxe2x89xa1Cxe2x80x94(C1-C12 alkyl) and xe2x80x94CH2xe2x95x90CH2xe2x80x94(C1-C12 alkyl) which may be cis- or trans- e.g. propenyl, butenyl, hexenyl, decenyl, and the like; propynyl, butynyl, hexynyl, undecynyl, and like alkynes.
Examples of acyl groups wherein R2 is a group represented by the formula:
are diphenyl ethers (Zxe2x95x90xe2x80x94Oxe2x80x94), diphenyl acetylenes (Zxe2x95x90xe2x80x94Cxe2x89xa1Cxe2x80x94), stilbenes (Zxe2x95x90xe2x80x94CHxe2x95x90CHxe2x80x94), and biphenyls (Z=a carbon to carbon bond). Among examples of such biphenyl groups, wherein Z is a carbon to carbon bond i.e. a phenyl to phenyl bond, are 4-[4-(butyloxy)phenyl]benzoyl, 4-[4-(cyclobutylmethoxy)-phenyl]benzoyl, 4-[4-cyclopentyl-methoxy)phenyl]benzoyl, 4-[4-(cyclohexylethoxy)-phenyl]benzoyl, 4-[4-(n-hexyloxy)-phenyl]benzoyl, 4-phenylbenzoyl, 4-[4-(11-amino-undecyloxy)-phenyl]benzoyl, 4-[4-(11-formamidoundecyloxy)phenyl]benzoyl, 4-[4-(iso-pentyloxy)phenyl]benzoyl, and the like. Examples of such diphenyl ether acyl groups R2 of the formula above wherein Z is an oxygen atom are 4-(4-butyloxyphenoxy)benzoyl, 4-(4-hexyloxyphenoxy)benzoyl, 4-(4-ethoxyphenoxy)benzoyl, 4-(4-benzyloxyphenoxy)benzoyl, 4-[4-(3-chlorobutyloxy)phenoxy]benzoyl, 4-(4-4decyloxyphenoxy)benzoyl, 4-[4-(3-di-methylaminopropoxy)phenoxy]benzoyl and the like. Examples of diphenylacetylene and stilbene acyl groups, R2, wherein Z is an acetylenic bond or an ethylene bond are 4-styrylbenzoyl, 4-(4-methoxystyryl)benzoyl, 4-(4-butyloxystyryl)benzoyl, 4-(phenylethynyl)benzoyl, 4-(4-ethoxyphenylethynyl)benzoyl, 4-(4-cyclohexyloxyphenyl-ethynyl)benzoyl, and the like. Examples of R2 acyl groups represented by the foregoing formula wherein Z is a carbon to carbon bond and R4 is represented by the formula xe2x80x94Oxe2x80x94(CH2)pxe2x80x2xe2x80x94Wxe2x80x94R5 are 4-[4-[2-(N-cyclohexylpiperidine-4-yl)ethoxy]phenyl]benzoyl, 4-[4-[2-(N-hexylpiperidine-4-yl)ethoxy]phenyl]benzoyl, 4-[4-[2-(4-benzylpiperidino)ethoxy]phenyl]benzoyl, 4-[4-[2-(4-cyclohexylpiperidino)ethoxy]phenyl]benzoyl and like diphenyl acyl groups. Examples of such acyl groups wherein R4 is represented by the formula xe2x80x94Yxe2x80x94R6 include 4-[4-(phenylethynyl)phenyl]benzoyl, 4-[4-(phenylethynyl)phenoxy]benzoyl, 4-[4-(hexynyl)phenyl]benzoyl, 4-[4-(styryl)phenoxy]benzoyl, 4-[4-4-benzylphenylethynyl)-phenyl]benzoyl, 4-[4-[4-[4-methylpiperidino)ethoxy]phenylethynyl]phenyl]benzoyl and like acyl groups. Such acyl groups wherein R4 is represented by the formula xe2x80x94Oxe2x80x94(CH2)pxe2x80x2xe2x80x94Wxe2x80x94R5 form salts of the basic amino groups of the piperidine and piperazine heterocyclic groups with both organic and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and with organic acids such as the sulfonic acids, benzenesulfonic acid, toluenesulfonic acid, methanesulfonic acid, acetic acid, chloroacetic acid, trifluoroacetic acid, benzoic acid, isophthalic acid, salicylic acid, citric acid, malic acid, succinic acid, malonic acid and like acids.
The following tables contain further examples of the cyclic peptides represented by the formula (1). Table 1 contains examples of cyclic peptides wherein the acyl group R2 is of the formula:
The following Table 2 illustrates the compound of the formula (1) wherein R2 is represented by the formula:
The following Table 3 illustrates compounds of formula 1 wherein R2 is of the formula as indicated from Table 2 and R4 is represented by the formula: xe2x80x94Oxe2x80x94(CH2)p-Wxe2x80x94R5.
The acyl cyclohexapeptides represented by formula (1) exhibit antiparasitic activity, for example, they are especially active against the infectious fungi Candida albicans and Candida parasilosis. They also exhibit significant activity against Aspergillus fumigatus. They are active both in vitro and in vivo and accordingly are useful in combating systemic fungal infections.
The compounds of the invention also inhibit the growth of certain organisms primarily responsible for opportunistic infections in immunosuppressed individuals. For example the compounds of the invention inhibit the growth of Pneumocystis carinii the causative organism of pneumocystis pneumonia in AIDS sufferers.
The antifungal activity of the compounds of the invention is determined in vitro in standard agar dilution tests and disc-diffusion tests wherein minimum inhibitory concentrations of the test compounds obtained. Standard in vivo tests in mice are used to determine the effective dose of the test compounds in controlling systemic fungal infections.
Tables 4A-E below contain the minimum inhibitory concentrations (MIC) in micrograms per milliliter (mcg/ml) for compounds of the invention against Candida albicans and Candida parapsilosis, and for certain compounds, the effective dose, ED50, in mice.
In Tables 4A-E, Rxe2x80x2xe2x95x90CH3, Rxe2x80x3xe2x95x90CH3, Rxe2x80x2xe2x80x3xe2x95x90CH3, Ryxe2x95x90OH, R7xe2x95x90OH and R1xe2x95x90H, In Tables 4A-D, Rxe2x95x90OH, while in Table E, Rxe2x95x90H.
In the Table 4A, R2 is of the formula:
with R3 being as indicated in the Table 4.
In Table 4B, R2 is of the formula:
where Z is xe2x80x94Oxe2x80x94 and R4 is as indicated.
Table 4C is as Table 4B, except Z is a carbon-carbon bond.
Table 4D indicates compound activities in which R2 is as defined.
In Table 4E, dideoxy (where Rxe2x95x90H) compounds are illustrated with R2 as indicated.
The non-dideoxy compounds of the invention (formula (1) are prepared with the amino nuclei of the cyclic hexapeptides which are represented by the formula when R2 is hydrogen. These amino nuclei are obtained from the known natural products by the known enzymatic deacylation by which the fatty acid side chains of the natural compounds are removed. For example, echinocandin B which can be represented by the formula (1) wherein Rxe2x80x2xe2x95x90Rxe2x80x3xe2x95x90Rxe2x80x3xe2x80x2xe2x95x90methyl, R is OH, Ry is hydroxy, R1 is H, R7 is OH, and R2 is linoleoyl, is deacylated to provide the echinocandin B nucleus (R2xe2x95x90H) with the deacylase produced by the organism Actinoplanes utahensis as described by U.S. Pat. Nos. 4,293,482 and 4,304,716.
The known natural cyclic hexapeptides which are N-deacylated to provide the amino nuclei starting materials include echinocandin B (also known as A-30912A), aculeacin (palmitoyl side chain), tetrahydoechinocandin B (stearoyl side chain), mulundocandin (branched C1s side chain), L-671,329 (C16 branched side chain), S 31794/F1 (tetradecanoyl side chain) , sporiofungin (C15 branched side chain) and FR901379 (palmitoyl side chain). The amino nuclei obtained by the N-deacylation are then acylated by employing known amino acylation procedures to provide the N-acyl cyclic hexapeptides represented by the formula (1) wherein R2 represents the acyl groups defined hereinabove. The acylating moiety is preferably an active ester of the carboxylic acid RCOOH such as the 2,4,5-trichlorophenyl ester. The R2COOH precursor acids are prepared by the hydrolysis of the nitrile R2CN or the ester R2COOC1-C4 alk. These nitrile and ester intermediates are prepared by known methods.
The alkoxy aromatic (ie. phenyl and biphenyl) compounds of Tables 5-10 are prepared by one of the two following procedures:
A. The hydroxyaromatic compound (1 equivalent) is dissolved in acetonitrile (200-300 ml) and a base, such as potassium t-butoxide or potassium carbonate,(1-equivalent), is added. An alkyl bromide, iodide, or p-toluenesulfonate (1 equivalent) is then added and the solution is refluxed for 6 hours. The solvent is evaporated in vacuo and the residue is dissolved in ether and 2N sodium hydroxide. The ether layer is dried over magnesium sulfate and evaporated to give the alkoxyaromatic product.
B. The hydroxyaromatic compound (1 equivalent), alkyl alcohol (1 equivalent), and triphenylphosphine (1 equivalent) are dissolved in tetrahydrofuran (200-300 ml) and diethylazodicarboxylate (1 equivalent) is added dropwise over 10 minutes at room temperature. After 17 hours the solvent is removed in vacuo and the residue is dissolved in ether. This organic layer is extracted with 2N sodium hydroxide solution, dried over magnesium sulfate, and evaporated to give a product which is crystallized from ether/pentane or, if the product contains a tertiary amine, the hydrochloride salt is formed and crystallized from methanol/ethyl acetate.
The alkynyl and alkenyl aromatic compounds contained in Tables 11-14 are prepared by the following procedure:
An aromatic bromide, iodide, or trifluoromethane-sulfonate (1 equivalent) is dissolved in acetonitrile (600 ml/0.1 mole of aromatic reactant) under a nitrogen atmosphere. An alkyne or alkene (1 equivalent), triethylamine (2 equivalents), palladium dichloride (0.05 equivalents), triphenylphosphine (0.1 equivalents), and cuprous iodide (0.025 equivalents) are added and the solution is refluxed for 17 hours. The solvent is removed in vacuo and the residue is slurried in ether (300 ml). Solids are removed by filtration and the filtrate is washed with 1N hydrochloric acid solution. The organic layer is dried over magnesium sulfate and evaporated to yield the product.
The aromatic boronic acids listed in Table 15 were prepared by the following procedure:
An aromatic halide (1 equivalent) is cooled to xe2x88x9278xc2x0 C. in tetrahydrofuran solvent. Butyl lithium (1.2 equivalents) is added. After 15 min triisopropyl borate (2 equivalents) is added and after 10 min of stirring the cooling bath is removed. When the reaction has warmed to room temperature water is added to quench the reaction followed by 1N HCl. The organic layer is removed under reduced pressure leaving a solid precipitate which is collected by filtration. This solid is washed with hexane leaving the pure boronic acid.
The terphenyl esters listed in Table 16 were made in the following manner:
An aromatic boronic acid (1 equivalent), methyl 4-iodobenzoate (1 equivalent), and potassium carbonate (1.5 equivalents) were mixed in a nitrogen-purged toluene solution. Alternatively, the trichloro phenyl ester of iodobenzoate my be used. Added tetrakis(triphenylphosphine)palladium (0.03 equivalents) and refluxed for 7 hrs. The solution was decanted to remove the potassium carbonate and reduced in vacuo. The residue was triturated with acetonitrile and the product solid was collected by filtration.
The aromatic nitrites or carboxylate esters described in Tables 5-16 can be converted to carboxylic acids by one of the two following hydrolysis procedures:
A. An aromatic nitrite is dissolved in ethanol and an excess of 50% sodium hydroxide solution and refluxed for 2 hours. Water is added until a solid precipitates. The precipitate is collected by filtration, added to dioxane and 6N hydrochloric acid solution and refluxed for 17 hours. Water is added and the carboxylic acid product crystallizes and is collected by filtration and dried under vacuum.
B. A carboxylate methyl ester is dissolved in methanol, excess 2N sodium hydroxide solution is added and the solution is refluxed for 5 hours. The solution is made acidic with excess hydrochloric acid and water is added until a precipitate forms. The carboxylic acid is collected by filtration and dried under vacuum.
The carboxylic acids are converted to 2,4,5-trichlorophenyl esters shown in Tables 17-25 by the following general procedure:
The aromatic acid (1 equivalent), 2,4,5-trichlorophenol (1 equivalent), and N,N-dicyclohexyl-carbodiimide (1 equivalent) are dissolved in methylene chloride. The mixture is stirred for 17 hours after which it is filtered. The filtrate is evaporated to dryness and the residue is dissolved in ether, filtered, and pentane is added until crystallization begins. The crystalline product is collected by filtration and dried under vacuum.
The dideoxy compounds of formula (1) are prepared by removing the benzylic and aminal hydroxy groups. The process includes subjecting a non-dideoxy compound of formula (1) (wherein R2 may be hydrogen or acyl) to a strong acid such as trichloroacetic acid, trifluoroacetic acid or borontrifluoride etherate with trifluoroacetic acid being preferred, and a reducing agent, such as sodium cyanoborohydride or triethylsilane, with triethylsilane being preferred. The reaction takes place at temperatures of between xe2x88x925 and 70xc2x0 C., and in a suitable solvent such as methylene chloride, chloroform or acetic acid, with dichloromethane being preferred. The acid should be present in an amount of 2 to 60 moles per mole of substrate, and the reducing agent should be present in an amount of 2 to 60 moles per mole of substrate. This process affords selective removal of the aminal and benzylic hydroxy groups.
The compounds represented by the formula (1) have improved properties over the previously known N-acyl hexapeptide antifungals. For example, in general the compounds exhibit oral bioavailability, a property which is important for any systemic antifungal agent. Also, numerous N-acyl compounds of the formula (1) have enhanced antifungal activity and enhanced water solubility.
Among the N-acyl hexapeptides represented by the formula (1) certain are preferred embodiments of the invention. The compounds wherein R2 is a diphenyl acyl group:
wherein Z is a carbon to carbon bond and R4 is an alkoxy, cycloalkoxy or cycloalkylalkoxy group are preferred antifungals. Also preferred compounds are represented when Z is a carbon to carbon bond and R4 is xe2x80x94Yxe2x80x94R6 and R6 is C1-C12 alkyl phenyl or substituted phenyl and Y is an acetylenic bond.
A further preferred group of N-acyl hexapeptides is represented when Z is a carbon to carbon bond and R4 is represented by xe2x80x94Oxe2x80x94(CH2)pxe2x80x94Wxe2x80x94Rs and wherein W is a piperidine group.
Examples of preferred compounds of the above first mentioned group include 4-(4-alkoxyphenyl)benzoyl wherein the alkoxy group is preferably a C5-C10 alkoxy group or C1-C4 alkoxy substituted by C3-C7 alkyl. Examples of such preferred compounds are represented by the formula 1 wherein R2 is 4-(4-n-hexyloxyphenyl)benzoyl, 4-(4-n-hexyloxyphenyl)benzoyl, 4-(4-n-octyloxyphenyl)benzoyl, 4-[4-(3,3-dimethylbutoxy)phenyl]benzoyl, 4-[4-(2-cyclopentyl-ethoxy)phenyl]benzoyl and 4-[4-(2-cyclohexyloxyethoxy)-phenyl]benzoyl.
Examples of the second above mentioned preferred compounds wherein R4 is xe2x80x94Yxe2x80x94R6 include 4-[4-(phenylethynyl)-phenyl]benzoyl and 4-[4-(n-butylethynyl)phenyl]benzoyl.
Examples of preferred compounds of the invention wherein R4 represents xe2x80x94Oxe2x80x94(CH2)pxe2x80x94Wxe2x80x94R5 are represented when R2 has the formula:
wherein W-R5 is piperidino, 4-n-propylpiperidino, 4-benzylpiperidino, 4-cyclohexylpiperidino, 4-cyclohexylmethylpiperidino, and the pharmaceutically acceptable acid addition salts such as the hydrochloride salts, the sulfate salts or the phosphate salts.
Preferred cyclohexylpeptide compounds are represented by the formula 1 wherein Rxe2x80x2xe2x95x90Rxe2x80x3=methyl, R1 is hydrogen and R2 is a preferred acyl group as defined hereinabove.
Table 26 is a list of the most preferred R2 substituents, wherein R=R7=Ry=OH; Rxe2x80x2xe2x95x90Rxe2x80x3xe2x95x90Rxe2x80x2xe2x80x3xe2x95x90CH3; and R1=H.
The N-acylhexapeptides provided by this invention are useful in the treatment of fungal infections both systemic infections and skin infections. Accordingly this invention also provides a method for treating fungal infections in man and animals which comprises administering to said host an antifungally effective non-toxic amount of an N-acyl-cyclohexapeptide represented by the formula 1. A preferred antifungal method comprises administering an N-acylhexapeptide compound where, in formula 1, Rxe2x80x2xe2x95x90Rxe2x80x2=methyl, R1 is hydrogen and R2 is a preferred acyl group as defined hereinabove.
The antifungal compound can be administered parenterally, e.g. i.m., i.p. or s.c., nasally, orally or can be applied topically for skin infections. The dose administered of course will vary depending on such factors as the nature and severity of the infection, the age and general health of the host and the tolerance of a particular host to the particular antifungal agent. The particular dose regimen likewise may vary according to such factors and may be given in a single daily dose or in multiple doses during the day. The regimen may last from about 2-3 days,up to about 2-3 weeks or longer.
This invention also provides pharmaceutical formulations useful for administering the antifungal compounds of the invention. These formulations comprise an N-acylhexapeptide represented by the formula 1 or a pharmaceutically acceptable, non-toxic salt thereof and a pharmaceutically acceptable carrier.
For parenteral administration the formulation comprises a compound of the formula 1 and a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose and other commonly used diluents. The formulation may contain a solubilizing agent such as a polyethylene glycol or polypropylene glycol or other known solubilizing agent. Such formulations may be made up in sterile vials containing the antifungal and excipient in a dry powder or lyophilized powder form. Prior to use, the physiologically acceptable diluent is added and the solution withdrawn via syringe for administration to the patient. For oral administration, the antifungal compound is filled into gelatin capsules or formed into tablets. Such tablets also contain a binding agent, a dispersant or other suitable excipients suitable for preparing a proper size tablet for the dosage and particular antifungal compound of the formula 1. For pediatric or geriatric use the antifungal compound may be formulated into a flavored liquid suspension, solution or emulsion. A preferred oral carrier system is lineolic acid, cremophor RH-60 and water and preferably in the amount (by volume) of 8% lineolic acid, 5% cremophor RH-60, and 87% sterile water. The compound is added to the system in an amount of 2.5 to 40 mg/ml.
For topical use the antifungal compound can be formulated with a dry powder for application to the skin surface or it may be formulated in a liquid formulation comprising a solubilizing aqueous liquid or non-aqueous liquid, e.g., an alcohol or glycol. Such formulations are useful forms for use in the antifungal method provided herein.
The N-acylcyclohexapeptides provided herein may be formulated as described above in unit dosage formu- lations comprising for injection between about 50 mg and about 500 mg per vial. For oral use gelatin capsules or tablets comprising between about 100 mg and about 500 mg per capsule or tablet can be provided.
Preferred formulations of the invention comprises the active ingredient presented by the formula 1 wherein Rxe2x80x2xe2x95x90Rxe2x80x3=methyl, R1 is hydrogen and R2 is 4-[4-(phenylethynyl)-phenyl]benzoyl in gelatin capsules or as active ingredient the antifungal represented by the formula 1 wherein Rxe2x80x2xe2x95x90Rxe2x80x3xe2x95x90methyl, R1 is hydrogen and R2 is 4-[4-[2-(4-cyclohexyl-piperidino)ethoxy]phenyl]benzoyl or the hydrochloride salt form thereof in tablet or gelatin capsules. Further preferred formulations are those in which a preferred compound, as described above, is employed.
In yet a further aspect of the present invention there is provided a method for treating patients suffering from Pneumocystis pneumonia. The method can be used prophylactically to prevent the onset of the infection which is caused by the organism Pneumocystis carinii. The N-acylcyclicpeptide can be administered parenterally, e.g. via intramuscular (i.m), intravenous (iv.) or intraperitoneal (i.p.) injection, or orally or by inhalation directly into the airways of the lungs. Preferably the cyclic peptide is administered via inhalation of an aerosol spray formulation of the compound.
An effective amount of a cyclic peptide will be between about 3 mg/kg of patient body weight to about 100 mg/kg. The amount administered may be in a single daily dose or multiple doses e.g. two, three or four times daily throughout the treatment regimen. The amount of the individual doses, the route of delivery, the frequency of dosing and the term of therapy will vary according to such factors as the intensity and extent of infection, the age and general health of the patient, the response of the patient to therapy and how well the patient tolerates the drug. It is known that PCP infections in AIDS patients are highly refractory owing to the nature of the infection. For example, in severe, advanced infections the lumenal surface of the air passages becomes clogged with infectious matter and extensive parasite development occurs in lung tissue. A patient with an advanced infection will accordingly require higher doses for longer periods of time. In contrast, immune deficient patients who are not severely infected and who are susceptible to PCP can be treated with lower and less frequent prophylactic doses.
The activity of the cyclicpeptide represented by the formula 1 is demonstrated in immunosuppressed rats. The tests were carried out in general as follows. One week after initiation of immunosuppression rats were inoculated intratracheally with parasites and maintained on immuno-suppression for the remainder of the study. Prophylactic treatments began one day after parasite inoculation and therapeutic treatments began 3 or 4 weeks later after moderate PCP developed. Eight or ten animals were assigned to the following groups: those receiving test compound; non-treated Pneumocystis infected control animals; animals treated with trimethoprim-sulfamethoxazole (TMP-SMX); or non-treated, non-infected control animals. The efficacy of different treatments was evaluated by monitoring animal weights and survival during the studies and by determining the severity of PCP at necropsy. Stained impression smears of the lungs and stained lung homogenates were evaluated to determine the intensity of P. carinii infection.
The immune deficient rats employed in the tests were prepared as follows. Female Lewis rats weighing from 120-140 g each were immune suppressed with methyl prednisolone acetate at a dose of 4 mg/100 g for the first week, 3 mg/100 g for the second week and continuing weekly thereafter at 2mg/100 g. All rats, except for the non-infected control rats, were inoculated intratracheally with 0.1 ml to 0.2 ml of Dulbecco""s Modified Eagle Media containing between  greater than 105 and 106 P. carinii (trophozoites, precysts and cysts) harvested from the lungs of heavily infected donor animals (infection scores of 6) and maintained as cryopreserved (liquid nitrogen) inocula. Rats were maintained on immune suppression and PCP was allowed to develop for 3 or 4 weeks before initiation of therapy with test compounds. Body weights were recorded weekly and rats were allocated into treatment groups such that each group had a similar distribution of percent weight loss among animals. Rats were treated with test compounds for 2 or 3 weeks and then were necropsied. For prophylaxis studies, administration of test compound was initiated one day after intratracheal inoculation of parasites and was continued until the rats were necropsied.
Following the evaluation period for test compounds, the rats were necropsied and test results evaluated by Giemsa-stained, silver-methenamine stained impression smears and/or by silver-methenamine stained lung homogenate (see below). Necropsy was carried out as follows. The test rats were anesthetized with a mixture of ketammine hydrochloride and xylazine and then exsanguinated via the right atrium. Internal organs in the abdominal and thoracic cavities were examined for gross lesions.
A small portion of lung tissue from the left lobe of each rat was used to make the impression smears described below. Giemsa-stained impression smears were evaluated to determine the total number of parasites (trophozites, precysts, and cysts). Impression smears from rats in groups whose treatments exhibited some anti-Pneumocystis activity (as judged by infection scores from Geisma-stained slides) and from rats in the control groups were also stained with methamine silver, a stain specific for the cyst wall of the organism. Impression smears were randomized, numbered, and then evaluated. The infection scores were used as follows:
A score of 6 was reserved for those infections with impression smears containing  greater than 1,000 organisms/field (too numerous to count). Giemsa-stained slides were examined microscopically using a final magnification of 1008xc3x97. Methenamine silver-stained slides were examined with a final magnification of 400xc3x97.
Cysts in rat lung tissue were quantified as follows. A small portion of lung tissue from the left lobe of each rat was used to make impression smears as described above. The remainder of each lung was weighed, placed in a tube containing Hanks balanced salt solution (HBSS) (40xc3x97 the lung weight) and homogenized using a Brinkman model tissue homogenizer. Two xcexcl samples of the homogenized lung samples (1:4 dilution in HBSS) were placed in wells of teflon-coated, 12-well slides, stained with methenamine silver, and the number of cysts were scored as described above for the impression smears.
The activity and efficacy of two preferred N- acylcyclohexapeptides in the test animals is presented below. The compound of the formula 1 wherein Rxe2x80x2xe2x95x90Rxe2x80x3=methyl, R1 is hydrogen and R2 is 4[(4-phenylethynyl)phenyl]benzoyl when administered as an aerosol solution at a concentration of 5 mg/ml for one hour, twice weekly for 5 weeks resulted in 90% reduction in P. carinii cysts in the lungs. When given orally at 10 mg/kg, bid for 3 weeks, the number of cysts in the lungs was reduced by  greater than 99% when compared with infected vehicle controls.
When the preferred N-acylcyclicpeptides were administered orally and by intraperitoneal injection the compound was effective in clearing P. carinii cysts from the lungs of heavily infected rats. For example, when the compound was administered at 10 or 40 mg/kg, bid for 4, 8 or 12 days, the number of identifiable cysts in the lungs of heavily infected rats was reduced by  greater than 99%. Similar efficacy was observed when the compound was administered i.p. at 1 mg/kg.
When tested orally for prophylactic activity, the preferred compound exhibited  greater than 99% cyst reduction in one of two studies when infected animals were dosed at 1 mg/kg and when given higher doses of 5 or 4 mg/kg.
Another preferred compound of the invention represented by the formula 1 wherein Rxe2x80x2xe2x95x90Rxe2x80x3xe2x95x90methyl, R1 is hydrogen and R2 is 4-[4-[2-(4-cyclohexylpiperidino)-ethoxy]phenyl]benzoyl as the hydrochloride salt was also effective in the treatment of PCP. Aerosol prophylaxis (two 60-minute treatments twice a week for 5 weeks) was highly effective. in preventing PCP in the infected immune suppressed rats. Aerosol therapy with 5, 10, 25, or 50 mg/ml of aerosolized solution reduced the number of cysts in the lungs by  less than 99% When compared to controls. Similar results were obtained by i.p. dosage.
The following examples of compounds of the invention and the manner of their preparation further describe the present invention.
The preparation of the derivatives of the A30912A nucleus was accomplished by the following general procedure, with Table 27 listing these derivatives.
The A30912A nucleus, prepared by methods known in the art from Aspergillus rugulosus (NRRL 8113;ATCC 58398) which provides the starting compound which is then deacylated using Actinoplanes utahensis (U.S. Pat. No. 4,293,482), with 2,4,5-trichlorophenol ester are dissolved dimethylformamide (25-50 ml) and stirred for 17-65 hours at room temperature. The solvent is removed in vacuo and the residue is slurried in ether and collected by filtration. The solid product is washed with methylene chloride and then dissolved in either methanol or acetonitrile/water (1:1 v/v). This solution is injected on a Waters 600E semi-preparative chromatography system using a Rainin Dynamax-60A C18 reverse-phase column. The column is eluted beginning with 20-40% aqueous acetonitrile and 0.5% monobasic ammonium phosphate (w/v) (monitored by UV at 230 nm and at a flow rate of 20 ml/min) until the unreacted A30912A nucleus is eluted and then deleting the buffer and eluting the product peak in aqueous acetonitrile. The fraction containing the product is evaporated in vacuo or lyophilized to provide the pure compound. The product may be analyzed by the same HPLC instrument using a Waters C18 Micro Bondapak column and eluting with 40% aqueous acetonitrile containing 0.5% monobasic ammonium phosphate (w/v) at a 2 ml/min flow rate and monitoring the UV at 230 nm. The products may also be analyzed by fast atom bombardment mass spectrometry (FABMS). (In the compounds used, Rxe2x80x2xe2x95x90Rxe2x80x3xe2x95x90Rxe2x80x2xe2x80x3xe2x95x90CH3, R1xe2x95x90H, Ry=OH, R1=H, R7=OH, and R2 is as defined).
Compounds such as those listed in Table 27 could be further modified at the phenolic hydroxy to provide R7=xe2x80x94OPO3HNa as shown in Table 28. The procedure is as follows:
The lipopeptide (1 equivalent) and tetrabenzylpyrophosphate (2 equivalents) were dissolved in dimethylformamide which had been dried over 13xc3x97 molecular sieves. Lithium hydroxide monohydrate (5 equivalents) was added and the stirred solution was monitored by HPLC. After 0.5 hr and 1 hr more lithium hydroxide (5 equivalents) was added. Between 1 and 2 hrs. the reaction was quenched with glacial acetic acid, the solvent removed under vacuum, and the residue purified over a semi-preparative C18 reverse-phase column using an aqueous acetonitrile eluent. The purified product was dissolved in (1/1) acetic acid/water with sodium acetate (1 equivalent) and 10% Pd/C catalyst. The solution was placed under an atmosphere of hydrogen gas and stirred for 1 hr. After filtering to remove the catalyst, the solution was lyophilized to provide the pure final product. The purity was assessed by analytical HPLC and the product was analyzed by fast atom bombardment mass spectrometry (RABMS).

Cyclic peptide antifungal agents and process for preparation thereof

Abstract: wherein

Abstract: Rxe2x80x3 and Rxe2x80x2xe2x80x3 are independently methyl or hydrogen;

Abstract: R2 is a acyl side chain. Also provided are formulations, methods of inhibiting fungal and parasitic activity, and a process for preparing dideoxy (Rxe2x95x90H) forms of the compounds.


In a process for the quantitative optical analysis of fluorescently labelled biological cells 5, a cell layer on a transparent support at the bottom 2 of a reaction vessel 1 is in contact with a solution 3 containing the fluorescent dye 4. The sensitivity of analytical detection can be considerably improved if to the fluorescent dye 4 already present in addition a masking dye 9, which absorbs the excitation light 6 for the fluorescent dye 4 and/or its emission light 7, is added to the solution 3 and/or if a separating layer 10 permeable to the solution and absorbing and/or reflecting the excitation light 6 or the emission light 7 is applied to the cell layer at the bottom 2. The separating layer 10 must be composed such that it has a high power of reflection for the luminescent light 11. Analogously, these process principles can also be used in receptor studies for the masking of the interfering background radiation in the quantitative optical analysis of fluorescently or luminescently labelled reaction components. In this case, a receptor layer 12 at the bottom 2 of a reaction vessel 1 is in contact with a solution in which a fluorescent or luminescent ligand 13 is dissolved. The sensitivity and accuracy of the analytical detection can be considerably improved here if a masking dye 9 which absorbs the excitation light 6 for the fluorescent dye and/or its emission light or the luminescent light is added to the supernatant 3. Instead of the masking dye in the solution 3 or optionally as an additional measure, a separating layer 10 permeable to the solution 3 and absorbing and/or reflecting the excitation light 6 and/or the emission light or the luminescent light can be applied to the cell or receptor layer 12 at the bottom 2.

The invention originates from a process for the quantitative optical analysis of fluorescently labelled biological cells which are in contact with a fluorescent dye solution or of luminescent cells which are applied to a transparent support at the bottom of a reaction vessel in the form of a coherent cell layer, or alternatively of fluorescently or luminescently labelled reaction components in a solution in which a fluorescent or luminescent ligand is dissolved, the solution being in contact with a receptor layer, which is specific for this ligand and situated on the transparent support at the bottom of the reaction vessel, whose fluorescent or luminescent radiation, which is characteristic of the receptor-ligand binding, is detected and analysed through the transparent bottom.
A problem in fluorescence measurement in biomedical assays is often that the fluorescence changes correlated with the biological cell action are small compared with the non-specific background fluorescence. As a result, the resolving power is greatly restricted. Conventional commercial measuring systems (fluorescence readers, Dynatech or SLT), cannot solve the problem, because owing to their optical measuring arrangement (excitation from xe2x80x98abovexe2x80x99 through the fluorescent liquid column of the supernatant) the signal can barely be detected in comparison with the background.
Apparatuses of newer construction (Labsystems), which illuminate the cells from the back through the transparent support of the reaction vessel, do have the advantage that on entry of the excitation light the cells are excited to fluorescence. Since the excitation light, however, enters further into the supernatant, which is also fluorescent, the fact that the non-specific background signal adulterates the cell signal cannot be avoided.
Even very complicated measuring systems (NovelTech, FLIPR: Fluorescence Imaging Plate Reader) are only able to decrease this background fluorescence using a special laser illumination geometry (excitation below about 45xc2x0). The reason for the failure of all problem-solving experiments on the measuring geometry is the fact that the actual cause of the background fluorescence cannot be decisively influenced hereby.
In the receptor binding studies carried out until now using fluorescently or luminescently labelled ligands, the labelled and unbound fraction in each case must be removed by processes like washing. Many coatings, however, are sensitive to these washing steps. Moreover, the removal of the unbound ligand is associated with a considerable outlay. The direct measurement of the receptor-ligand association or dissociation is not possible in this process.
The invention is based on the object of improving the sensitivity of the optical analysis of fluorescently labelled or luminescent cells in a cellular assay in order to be able to measure, for example, membrane potential changes which are as low as possible on the basis of fluorescence changes of potential-sensitive dyes. In this case, the sensitivity of the measuring system should be so high that potential changes of below 5 mV can be detected at least qualitatively. In the case of luminescent cells, an increase in the detection of the luminescence signal should be achieved. Moreover, the method should be suitable for screening with a high sample throughput.
The invention is furthermore based on the object of simplifying receptor binding studies based on fluorescently or luminescently labelled ligands or receptors and making possible continuous measurement of the receptor binding interaction (kinetics). Owing to the reduction in the process steps necessary, this method should be particularly suitable for screening with a high throughput and for diagnostic applications.
It was only possible to achieve the required high resolution with low membrane potential changes after it was possible to eliminate the cause of the interfering overlapping of the non-specific background fluorescence and the specific fluoresence of the cells. The process according to the invention developed for this purpose is based on the fundamentally new idea of masking the excitation energy and the fluorescence not originating from the biological object. To do this, in addition to the fluorescent dye, a further dye is added which completely absorbs the excitation light of the fluorescent dye and/or its emission light without affecting the fluorescence of the cells. By means of this absorption, the non-specific background signal is masked and the useful cell signal can be detected with a resolution which was previously not possible.
An alternative solution which is within the scope of the invention is that a separating layer, which is permeable to the solution and which absorbs and/or reflects the excitation light for the fluorescent dye and/or its emission light without adversely affecting the cell properties, is applied to the cell layer. At the same time, the thickness of the separating layer is selected such that fluorescence is no longer detectable in the dissolving mixture with the fluorescent dye but without the cells.
A further variant of the invention is that the method of the separating layer according to the invention is also used for increasing the sensitivity in the quantitative optical analysis of luminescent (luminous) biological cells which are applied to a transparent support in the form of a coherent cell layer. For this purpose, the optical properties of the separating layer permeable to the solution are selected such that it reflects the luminescent light as strongly as possible without adversely affecting the cell properties. In this manner, it is possible to increase the luminescence intensity and thus the measured effect considerably.
The process according to the invention can be used in a completely analogous manner for the quantitative optical analysis of fluorescently or luminescently labelled reaction components in a reaction vessel filled with a solution, the fluorescent or luminescent ligand being present in dissolved form and the solution being in contact with a receptor layer which is specific for this ligand, applied to a transparent support at the bottom of the reaction vessel or deposited thereon, whose fluorescent or luminescent radiation, which is characteristic for receptor-ligand binding, is detected and analysed through the transparent bottom. In this case, the solution according to the invention of the object described above is based on the fact that the free ligand which is in the supernatant, i.e. in solution, and its non-specific fluorescence or luminescence is masked by an additional dye and/or by a diffusely absorbing or reflecting separating layer and thus the cause of the interfering overlapping of the non-specific background fluorescence and the specific fluorescence of the ligand in the solution is eliminated. Since the non-bound ligand is masked in this manner, the measured fluorescence or luminescence is a direct measure of the ligand-receptor interaction. It can be measured directly in this process with time resolution.
In receptor studies, in analogy to the process described above, the invention thus relates to a process variant in which a masking dye is added to the solution and/or a separating layer permeable to the solution is applied to the receptor layer, the optical properties of the masking dye and/or of the separating layer being selected such that the excitation light for the fluorescent dye of the ligand present in the solution and/or its emission light or its luminescent light is absorbed by the solution or the separating layer or reflected at the separating layer. In this case, the thickness of the separating layer is selected such that fluorescence is no longer detectable in the dissolving mixture with the fluorescent dye, but without the receptor layer.
The separating layer preferably consists of polymeric latex beads (e.g. polystyrene, polyurethane, butadiene, acrylonitrile). The latex beads can also be dyed with a masking dye, which in this case must have an adequately high polymer dyeing capacity.
In the first-mentioned process, the masking dye should be as well distributed as possible in the solution which also contains the fluorescent dye in dissolved form. Since, as a rule, the solvent is water, a masking dye is expediently employed which possesses good water solubility ( greater than 2 g/ml) and has no cytotoxic side effects.
According to a further development of the invention, after the replacement of the supernatant containing a fluorescent dye by a fluorescent dye-free solution, a further masking dye is added which suppresses a non-specific fluorescence on the reaction vessel wall.
The following advantages are achieved using the invention: The new process described is not tied to a certain measuring system, but can be used, because it is not a specifically technical solution, by many commercially available apparatuses. These include virtually all fluorescence readers which can illuminate and also measure transparent reaction vessels, e.g. microtitre plates, from the bottom. With a very low outlay (minimal additional costs only for the special absorption dyes), it is possible for the first time by this means to advance in a resolving area, e.g. in the measurement of potential changes in cell membranes by measurement of the change in fluorescence of potential-sensitive fluorescent dyes, which was unachieved until now. For the first time it is possible even in the case of very low changes to carry out a direct comparison of the results from various reaction vessels (e.g. various wells in a microtitre plate), such that the complicated procedure of the determination of the relative change in a reaction vessel can be dispensed with. As a result the number of measurements to be determined, e.g. for kinetic measurements, decreases. The outlay in terms of time for a measuring programme is markedly reduced and the possibility created of obtaining identical results by a simple individual measurement (e.g. end point determination) with the use of reference to a separate control batch. The uniformity of the biological batch required in this case (e.g. homogeneous cell layer) is generally afforded, for example, for microtitre plates.
Surprisingly, the use of various water-soluble dyes and also their mixtures in the very different cells tested showed no negative effect on the physiology of the cells (e.g. reaction of the cells in comparison with electrophysiological measurements such as whole-cell patch-clamp, or effects of the pharmaceuticals investigated). The use of undissolved dye pigments or inorganic finely divided particles was also surprisingly well tolerated by the biological objects.
As a result of the simple process described for masking the background fluorescence in quantitative fluorescence measurement in biomedical assays, connected with an increase in the sensitivity, e.g. when using potential-sensitive fluorescent dyes, and the adaptability of this process, e.g. to microtitre plates as reaction vessels, the use of such measuring techniques will significantly simplify high-throughput screening, especially as no increased technical expenditure is necessary for the realization of the advantages outlined, but existing commercial measuring apparatuses are sufficient for this purpose. In receptor-ligand studies, the advantage essential to the invention is that, on account of the masking of the non-specific fluorescence or luminescence, it is no longer necessary to remove the unbound fraction of the ligands. As a result, the test procedures are considerably simplified, damage to and destruction of the sensitive coatings or of the biological objects such as, for example, cells are avoided and the sensitivity and thus also the accuracy of the measurement are improved. As a result of the use of microparticles, the utilizable surface area for the coating of fluorescently or luminescently labelled ligands can be significantly increased. By means of suitable measures, e.g. relatively high specific density or the use of magnetizable particles, the settlement and concentration of the microparticles on the transparent support can be achieved. In this case too, the fluorescence or luminescence of the unbound ligands in the supernatant is effectively suppressed by the masking.
Since the interaction between the ligands and the receptor must not be interrupted by the removal of the unbound fraction, a continuous measurement of the interaction between ligand and receptor (kinetics) can be carried out in this manner even in an individual reaction batch.

Masking background fluorescence and luminescence in optical analysis of biomedical assays